Methods for identifying human cell lines useful for endogenous gene activation, isolated human lines identified thereby, and uses thereof

ABSTRACT

The invention concerns human cells which, due to an activation of the endogenous human EPO gene, are able to produce EPO in an adequate quantity and purity to enable a cost-effective production of human EPO as a pharmaceutical preparation. Furthermore the invention concerns a process for the production of such human EPO-producing cells, DNA constructs for the activation of the endogenous EPO gene in human cells as well as a process for the large-scale production of EPO in human cells.

FIELD OF THE INVENTION

The invention relates to methods for identifying human cells useful inendogenous gene activation, in order to produce human proteins. Theinvention also involves processes for manufacture of proteins, such ashuman proteins in cells identified in this manner, as well as theisolated cells so identified.

BACKGROUND AND PRIOR ART

The production of human proteins by endogenous gene activation in ahuman cell line is known. See, e.g., WO 93/09222, WO 94/12650 and WO95/31560 for example, describing the production of human erythropoietinand other human proteins in human cell lines by endogenous geneactivation.

These references do not advise, however, as to what criteria have to beobserved when selecting cells used to produce human proteins. In fact,the methods described in these references do not ensure high yield andcontaminant free production of human protein. In fact, only low yieldsof human proteins are achieved following the above cited references.

As noted, supra, human erythropoietin is described in these referencesas a protein, the production of which is desired. A discussion oferythropoietin and the art relating to its production is set forth here,although it is to be borne in mind that erythropoietin production issimply exemplary of the invention, which is in no way limited to thisprotein.

Erythropoietin (“EPO” hereafter) is a glycoprotein which stimulates theproduction of red blood cells. EPO is present only in very lowconcentrations in the blood plasma of healthy persons, so it is notpossible to prepare large amounts via purification of plasma. EP-B-0148605 and EP-B-0205 564, incorporated by reference, describe theproduction of recombinant human EPO in Chinese Hamster ovary, or “CHO”cells. The EPO described in EP-B-0148 605 has a higher molecular weightthan EPO purified from urine and is not O-glycosylated. The EPO from CHOcells described in EP-B-0 205 564 is available in large amounts and inpure form, but it is derived from non-human cells. Further, theproduction yield of CHO cells is also often relatively limited.

As alluded to supra, it is known that human EPO (“hEPO”) can be isolatedfrom the urine of patients with aplastic anemia (Miyake et al., J. Biol.Chem. 252 (1977), 5558-5564). A seven-step process is disclosed in thisreference, which involves, inter alia, ion exchange chromatography,ethanol precipitation, gel filtration and adsorption chromatography. Inthis process an EPO preparation with a specific activity of ca. 70,000U/mg protein is obtained in a 21% yield. The disadvantages of thisprocess and other processes for isolating urinary EPO include obtainingthe starting material in adequate amounts and with a reproduciblequality. Furthermore, the purification of hEPO from urine is difficultand even a purified product is not free of urinary impurities.

GB-A-2085 887, incorporated by reference, describes a process for theproduction of human lymphoblastoid cells which are able to produce EPOin small amounts. It is not possible to economically produce EPO of thedesired quality using the human lymphoblastoid cells disclosed herein.

WO 91/06667 as noted supra, describes a process for the recombinantproduction of EPO. In this process the endogenous EPO gene isoperatively linked to a viral promoter in a first process step byhomologous recombination, in primary human embryonic kidney cells. Therecombined DNA is then isolated from these cells. In a second step, theisolated DNA is transformed into CHO cells, and the expression of EPO inthese cells is analyzed. There is no indication that it is possible toproduce EPO in human cells.

WO 93/09222 describes the production of EPO in human cells. In thisprocess relatively high levels of EPO production, i.e., up to 960,620mU/10⁶ cells/24 hours is achieved using human fibroblasts which havebeen transfected with a vector containing the complete EPO gene. Thesetransfected cells contain an exogenous EPO gene which is not located atthe correct EPO gene locus, leading to problems with respect to thestability of the cell line. The reference does not discuss constitutiveEPO production. Moreover, there is also no information about whether theEPO produced is of sufficient quality for, e.g., pharmaceutical use.

Activation of the endogenous EPO gene in human HT1080 cells is alsodescribed in this reference, but production of only 2,500 mU/10⁶cells/24 hours (corresponding to ca. 16 ng/10⁶ cells/24 hours) is found.Such low production levels are unsuitable for economic production of apharmaceutical preparation.

WO 94/12650 and WO 95/31560, incorporated by reference, describe that ahuman cell with an endogenous EPO gene activated by a viral promoter iscapable, after amplification of the endogenous EPO gene, of producingEPO in an amount of up to ca. 100,000 mU/10⁶ cells/24 hours(corresponding to ca. 0.6 μg/10⁶ cells/24 hours). Even this amount isstill not sufficient for the economic production of a pharmaceuticalpreparation.

As indicated, supra, the cells and cell lines disclosed in theliterature relating to endogenous gene activation, while useful, are byno means totally satisfactory. It has now been found, however, that itis possible to identify and to isolate cells and cell lines which willbe useful in high yield production of proteins, following endogenousgene activation via, e.g., homologous recombination. Hence, one aspectof the invention is a method for identifying such cells and cell lines.A second feature of the invention are the cells and cell lines soidentified. Yet a third feature of the invention is the use of the cellsand cell lines for the production of proteins, using these cells andcell lines. How these and other aspects of the invention are achievedwill be seen from the disclosure which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic representation of the amplification of homologyregions of the EPO gene from the region of the 5′ untranslatedsequences, exon 1 and intron 1 and the primers of PCR product 1 (upperstrand=SEQ ID NO: 1; lower strand=SEQ ID NO: 2) and PCR product 2 (upperstrand=SEQ ID NO: 3; lower strand=SEQ ID NO: 4);

FIG. 2 shows a schematic representation of a plasmid which contains EPOhomology regions from the region of the 5′ untranslated sequences, exon1 and intron 1;

FIG. 3 shows a schematic representation of a gene activation sequencewhich contains the Rous-sarcoma virus promoter (RSV), the neomycinphosphotransferase gene (NEO), the early polyadenylation region of SV40(SVI pA), the early SV40 promoter (SVI), the dihydrofolate reductasegene (DHFR), an additional early SV40 polyadenylation region and thecytomegalovirus immediate-early promoter and enhancer (MCMV);

FIG. 4a shows the construction of the EPO gene targeting vector p176;

FIG. 4b shows the construction of the EPO gene targeting vectors p179and p187;

FIG. 4c shows the construction of the EPO gene targeting vector p189(DSM 11661);

FIG. 4d shows the construction of the EPO gene targeting vector p190;

FIG. 4e shows the construction of the EPO gene targeting vector p192;

FIG. 5 shows a schematic representation of the construction of EPO cDNAwith signal sequence mutations;

FIG. 6a shows the hybridization of cellular DNA with a probe from theCMV region of the gene cassette shown in FIG. 3; lanes 1 to 4 each showDNA from human cells, cleaved with the restriction enzymes AgeI andAscI; lane 1: EPO-producing HeLa S3 cell amplified with 1,000 nMmethotrexate (MTX); lane 2: EPO-producing HeLa S3 cell amplified with500 nM MTX; lane 3: EPO-producing HeLa S3 cell without amplification;lane 4: HeLa S3 cell without activated EPO gene; lane 5:digoxigenin-labelled length marker. The size of the hybridizing fragmentin lanes 1 to 3 is ca. 5,200 bp; and

FIG. 6b shows the hybridization of a probe from the coding region of EPOwith DNA of human cells. Lane 1 shows digoxigenin-labelled lengthmarkers; lanes 2 to 4 show DNA from human cells, cleaved with therestriction enzymes BamHI, HindIII and SalI; lane 2: EPO producing HeLaS3 cell amplified with 500 nM MTX (length of the band produced by thenon-activated endogenous gene: 3,200 bp; length of the copy of the EPOgene activated by gene targeting: 2,600 bp); lane 3: DNA from anon-amplified EPO-producing HeLa S3 cell; lane 4: DNA from a HeLa S3control cell.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As indicated, supra, one feature of this invention is a method foridentifying and isolating cells and cell lines, preferably human cellsand cell lines, which are useful in the production of endogenousproteins via gene activation, preferably by homologous recombination. Inthe method, a cell or cell line, preferably human, is first tested todetermine if the desired gene of interest, which will generallycorrespond to the naturally occurring nucleotide sequence, is present.The details of how this is determine and are set forth, infra. If it isdetermined that the cell or cell line contains the desired sequence,then it is determined if a population of the cell or cell line doubles,at least five times, in a period of 14 days, when incubated insuspension culture. If the cell or cell line satisfies this criterion,then it is tested to see if a population of the cell or cell line willdouble, at least five times over 14 days if grown in serum free culturemedium. If the cell or cell line satisfies this criterion, then it willbe useful in activation of the gene of interence, via, e.g., homologousrecombination.

It is preferred that the starting materials are immortalized human celllines. These are preferred because they have known advantages withrespect to culturability which need not be summarized here. Such celllines, it must be borne in mind, can exhibit mutations in their genomes,so the presence of the desired sequence must be confirmed. Thepolymerase chain reaction, or “PCR,” for example, can be used todetermine this, following standard protocols.

Assuming the cell or cell line possesses the desired sequence, it istested for its ability to be cultured. Suspended cells are easier toferment and the fermentation can be more easily adapted to largerdimensions, such as large fermenters with a volume of 10 to 50,000liters. Consequently, the selected cells should either be cells whichare known to be culturable in suspension or they should be readilyadaptable to suspension culture. One determines this by culturing thecells for 14 days while stirring continuously. If the population of thecells doubles at least five times within this period, they are regardedas being suitable for the next step of the invention. The number ofpopulation doublings can be determined by periodic determinations of thecell count, e.g., by mechanical cell counting or by measuring theoptical density of the cell suspension.

A further important feature of the cells or cell lines of the inventionis the culturability in serum-free medium. Since the purification ofproteins from serum-free cell cultures is considerably simpler, and inserum-free culture there is no risk of contamination with animalpathogens such as viruses, it should be possible to culture the selectedcells in serum-free culture. To determine this, the cells are culturedfor 14 days at a density of 1 to 10×10⁵ cells per ml in culture vesselscontaining serum-free medium (e.g., RPMI 1640 containing insulin,transferrin and selenide). If the population of the cells doubles atleast five times during this culture period, determined as above, theyare regarded as being suitable for serum-free culture.

A further important feature of the invention, representing a preferredembodiment, is the generation time. The selected cells should exhibit ahigh proliferation in media such as DMEM, 10% fetal calf serum or RPMI1640 containing 10% fetal calf serum, i.e., within one week in culturetheir population should double 10 to 256 times, preferably 64 to 128times. To determine this, the cells are seeded in culture plates at aconcentration of 0.1 to 10×10⁵ cells per ml, preferably 0.5 to 2×10⁵cells per ml and the cell count is determined every two to three dayswith the aid of a cell counting chamber after or without trypsinization.Cells which have a sufficiently short generation time are particularlysuitable for large-scale production of human proteins by endogenous geneactivation.

A further preferred embodiment of the invention is the absence ofdetectable endogenous expression, i.e., transcription and translation,of the target gene. Preferably, those cell lines are selected forendogenous gene activation which have essentially no or no endogenousexpression of the target gene. To determine this, the cells are seededat a cell density of 0.01 to 2×10⁶ cells/ml, preferably 0.5 to 1×10⁶cells/ml of culture medium. After a predetermined time period, e.g., 24hours, the cell supernatant is removed, the cells are discarded and thecontent of the target protein is determined in the cell supernatant bymeans of known test procedures, e.g., ELISA. In the case of EPO, thedetection limit may be 10 pg/EPO/ml. Cells which yield less than 10 pgprotein when seeded at 10⁵ cell/ml are regarded as non-producers and areparticularly suitable.

It is especially preferred that the target gene exhibit polysomy in theselected cell. The presence of more than two chromosomal copies of thetarget gene in the cell significantly increases the yield of protein,following homologous recombination. The cells Namalwa (Nadkarni et al,Cancer 23 (1969), 64-79) or HeLa S3 (Puck et al., J. Exp. Med. 103(1956), 273-284) which possess three copies of chromosome 7 have provento be particularly suitable for the production of EPO, whose gene lieson chromosome 7. Further examples of cell lines that contain more thanone copy of chromosome 7 are the colon adenocarcinoma cell line SW-480(ATCC CCL-288; Leibovitz et al., Cancer Res. 36 (1976), 4562-4567), themalignant melanoma cell line SK-MEL-3 (ATCC HTB 69; Fogh and Tremp, in:Human Tumor Cells in vitro, pp 115-159, J. Fogh (ed.), Plenum Press, NewYork 1975), the colon adenocarcinoma cell Colo-320 (ATCC CCL-220; Quinnet al., Cancer Res. 39 (1979), 4914-4924)), the melanoma cell lineMEL-HO (DSM ACC 62; Holzmann et al., Int. J. Cancer 41 (1988), 542-547)and the kidney carcinoma cell line A-498 (DSM ACC 55; Giard et al., J.Natl. Cancer Inst. 51 (1973), 1417-1423).

The number of chromosomes in the genome of a cell line can be determinedby using DNA probes which are specific for the respective chromosomeor/and the locus of the target gene.

It is especially preferred that the cell line used for endogenous geneactivation correctly glycosylates the desired target protein. A humancell line which synthesizes the target protein with a glycosylationpattern which is comparable to and is preferably indistinguishable fromthe naturally occurring target protein, especially in the number ofsialic acid residues is particularly preferred. The ability to correctlyglycosylate can be tested by transiently transfecting the cell ofinterest with, e.g., a vector such as an extrachromosomal vector whichcontains the desired target gene under the control of a promoter that isactive in the cell. After transient expression of the target gene thecell supernatant or/and the cell lysate is analyzed by isoelectricfocusing. The presence of correct glycosylation can be easilydetermined. For example, non-glycosylated EPO, i.e., recombinant EPOfrom E. coli cells, has activity comparable to glycosylated EPO in invitro experiments. However, in in vivo experiments, non-glycosylated EPOis considerably less effective. In order to determine whether a startingcell line is able to produce EPO with correct glycosylation, comparisoncan be made to urinary EPO, or with recombinant EPO from CHO cells,which is known to have a form of glycosylation which is active in humansand whose glycosylation is substantially identical to that of urinaryEPO. Glycosylation is preferably compared by isoelectric focusing.

It is preferred that the cell line be free of infectious contamination,i.e., infectious viral particles or mycoplasmas. The examination for thepresence of viral contamination can be carried out by means of cellculture, in vivo analyses or/and detection of specific viral proteins.

Cells or cell lines so identified can be used in a process for theproduction of human proteins by endogenous gene activation of a humancell line, which meets the above listed criteria.

The process according to the invention can be used to produce substancessuch as EPO, thrombopoietin (TPO), colony-stimulating factors such asG-CSF or GM-CSF, proteins which influence blood coagulation such ast-PA, interferons such as IFN-α, IFN-β or IFN-γ, interleukins such asIL-1 to IL-18, chemokines such as MIP, neurotrophic factors such as NGFor BDNF, proteins which influence bone growth such as IFG-BPs, hedgehogproteins, tumor growth factors such as TFG-β, growth hormones such ashGH, ACTH, enkephalins, endorphins, receptors such as interleukin orinsulin receptors in soluble or/and membrane-bound forms and otherprotein binding proteins. The process is particularly preferably used toproduce EPO.

Endogenous gene activation can be carried out according to knownmethods. Preferably, it comprises the steps:

(a) providing human starting cell lines which contain at least one copyof an endogenous target gene with the desired nucleic acid sequence andwhich have been identified as being suitable for the expression of thetarget gene via the processes described supra,

(b) transfecting the cells with a DNA construct comprising:

(i) two flanking DNA sequences which are homologous to regions of thetarget gene locus in order to allow homologous recombination,

(ii) a positive selection marker gene,

(iii) optionally a negative selection marker gene,

(iiii) optionally an amplification gene and

(v) a heterologous expression control sequence which is active in thehuman cell,

(c) culturing the transfected cells under conditions in which aselection takes place for the presence of the positive selection markergene and optionally for the absence of the negative selection markergene,

(d) analyzing the cells that can be selected according to step (c),

(e) identifying the cells producing the desired target protein and

(f) optionally amplifying of the target gene in the selected cells.

The DNA construct used to produce the cell producing the desired humanprotein contains two flanking DNA sequences homologous to regions of thetarget gene locus. Suitable flanking sequences can be selected for inaccordance with the methods described in WO 90/11354 and WO 91/09955.The flanking sequences preferably are at least 150 bp long. Thehomologous DNA sequences are particularly preferably selected from the5′ region of the target gene, e.g., 5′-untranslated sequences,signal-sequence-coding exons and introns located in this region, e.g.,exon 1 and intron 1.

The positive selection marker gene can be any selection marker genesuitable for eukaryotic cells which leads to a selectable phenotype whenexpressed. Antibiotic resistance, auxotrophy etc. are examples of this.A particularly preferred positive selection marker gene is the neomycinphosphotransferase gene.

A negative selection marker gene can be present, but is not required.Examples of these include HSV thymidine kinase gene, expression of whichleads to death in the presence of a selection agent. The negativeselection marker gene is located outside of the two flanking homologoussequence regions, and is preferably downstream of the 3′ homologyregion.

If amplification of the endogenously activated target gene in the humancell is desired, the DNA construct is provided with an amplificationgene. Examples of suitable amplification genes are the dihydrofolatereductase gene, the adenosine deaminase gene, the ornithinedecarboxylase gene, etc. A particularly preferred amplification gene isthe dihydrofolate reductase gene, more particularly a gene coding for adihydrofolate reductase arginine mutant which has higher sensitivity forthe selective agent (methotrexate) than the wild-type gene. See Simonsenet al., Proc. Natl. Acad. Sci. USA 80 (1983), 2495, incorporated byreference.

It is especially preferred that the DNA construct used for theendogenous gene activation contains a heterologous expression controlsequence that is active in a human cell. The expression control sequencecomprises at least a promoter and preferably further sequences whichimprove expression, such as enhancer sequences. The promoter can be aregulatable or constitutive promoter. The promoter is preferably astrong viral promoter, e.g., an SV40 or a CMV promoter. The CMVpromoter/enhancer is particularly preferred.

Endogenous genes as used herein refers to an endogenous gene notmodified in the region which encodes the desired, mature polypeptide.

In accordance with the invention, one can isolate a human cell whichcontains a copy of an endogenous EPO gene in operative linkage with aheterologous expression control sequence that is active in the humancell and which is capable of producing at least 200,ng EPO/10⁶ cells/24hours without prior gene amplification. Such human cells preferablyproduce 200 to 3000 ng EPO/10⁶ cells/24 hours and are most preferablyable to produce 1000 to 3000 ng EPO/10⁶ cells/24 hours, underappropriate culture conditions.

Human cells which contain several copies of an endogenous EPO gene, eachin operative linkage with a heterologous expression control sequencethat is active in the human cell and are able to produce at least 1000ng EPO/10⁶ cells/24 hours, are also a facet of the invention. Such humancell lines preferably produce 1,000 to 25,000 ng EPO/10⁶ cells/24 hoursand most produce 5,000 to 25,000 ng EPO/10⁶ cells/24 hours underappropriate culture conditions.

The human cells and cell lines of the invention can be of any cell type,provided that they are culturable in vitro, in serum free medium,particularly in suspension culture. In this manner, it is possible toproduce EPO in large fermenters of about 1,000 liters or more.

Preferably, immortalized cells such as HT 1080 cells (Rasheed et al.,Cancer 33 (1974), 1027-1033), HeLa S3 cells (Puck et al., J. Exp. Med.103 (1956), 273-284), Namalwa cells (Nadkarni et al., Cancer 23 (1969),64-79) or a cell derived therefrom are used. HT1080 cells, HeLa S3cells, and cells derived therefrom are especially preferred.

The cells of the invention are characterized by linkage of theendogenous EPO gene to a heterologous expression control sequence thatis active in the human cell. The expression control sequence comprises apromoter, and preferably, further sequences that improve expression,such as enhancer sequences. The promoter can be a promoter that can beregulated, or a constitutive promoter. The promoter is preferably astrong viral promoter, e.g., an SV40 promoter, or a CMV promoter. TheCMV promoter/enhancer is particularly preferred.

Furthermore, in order to optimize expression of proteins such as EPO, itis preferable that the endogenous gene in the human cell that is inoperative linkage with the heterologous promoter have a signal peptidecoding sequence which is different from the naturalsignal-peptide-coding sequence, and preferably codes for a signalpeptide with a modified amino acid sequence. A signal-peptide-codingsequence which codes for a signal peptide sequence that is modified inthe region of the four first amino acids. These first four amino acidssatisfy the formula:

Met Xaa Xaa Xaa (SEQ ID NO: 5)

wherein the first Xaa is Gly or Ser, the second Xaa is Ala, Val, Leu,Ile, Ser or Pro, and the third Xaa is Pro, Arg, Cys, or His, with theproviso that this four amino acid sequence is not

Met Gly Val His (SEQ ID NO: 6).

Particularly preferred are:

(a) Met-Gly-Ala-His (SEQ ID NO: 7),

(b) Met-Ser-Ala-His (SEQ ID NO: 8),

(c) Met-Gly-Val-Pro (SEQ ID NO: 9) or

(d) Met-Ser-Val-His (SEQ ID NO: 10)

The sequence of the first four amino acids of the signal peptide isespecially preferably Met-Ser-Ala-His.

A further aspect of the present invention is a DNA construct useful foractivating an endogenous EPO gene in a human cell comprising:

(i) two flanking DNA sequences which are homologous to regions of thehuman EPO gene locus, selected from 5′ untranslated sequences, exon 1and intron 1, in order to allow homologous recombination wherein amodified sequence is present in the region of exon which codes for afour amino acid sequence as described supra:

(ii) a positive selection marker gene,

(iii) a heterologous expression control sequence which is active in ahuman cell and

(iv) optionally an amplification gene.

A further aspect of the present invention is a DNA construct foractivating an endogenous EPO gene in a human cell comprising:

(i) two flanking DNA sequences which are homologous to regions of thehuman EPO gene locus and are selected from 5′ untranslated sequences,exon 1 and intron 1 in order to allow a homologous recombination,

(ii) a positive selection marker gene,

(iii) a heterologous expression control sequence which is active in ahuman cell wherein the distance between the heterologous expressioncontrol sequence and the translation start of the EPO gene is not morethan 1100 bp and

(iv) optionally an amplification gene.

Surprisingly it has been found that when the EPO signal sequence ismodified and/or when the distance between the heterologous expressioncontrol sequence and the translation start of the EPO gene is shortened,optimized expression is obtained. The distance between the promoter ofthe heterologous expression control sequence and the translation startof the EPO gene is preferably not more than 1100 bp, particularlypreferably not more than 150 bp and most preferably not more than 100bp. A particularly preferred example of a DNA construct that can be usedaccording to the invention is the plasmid p189 (DSM 11661) or a plasmidderived therefrom.

Yet a further aspect of the present invention is a process for theproduction of human EPO in which a human cell according to the inventionis cultured in a suitable medium under conditions in which production ofEPO takes place and the EPO is isolated from the culture medium. Aserum-free medium is preferably used as the medium. The cells arepreferably cultured in suspension. The production preferably takes placein a fermenter in particular in a large fermenter with a volume of forexample 10-50,000 liters.

The isolation of human EPO from the culture medium of human cell linespreferably comprises the following steps:

(a) passing the cell culture supernatant over an affinity chromatographymedium and isolating the fractions containing EPO,

(b) optionally passing the fractions containing EPO over a hydrophobicinteraction chromatography medium and isolating the fractions containingEPO,

(c) passing the fractions containing EPO over hydroxy-apatite andisolating the fractions containing the EPO and

(d) concentrating and/or passing over a reverse phase (RP)-HPLC medium.

Step (a) of the purification process comprises passing the cell culturesupernatant, which can optionally be pretreated, over an affinitychromatography medium.

Preferred affinity chromatography media are those on which a blue dye iscoupled. A particularly preferred example is blue-Sepharose. Afterelution from the affinity chromatography medium the eluate containingEPO is optionally passed over a hydrophobic interaction chromatographymedium. This step is expedient if a culture medium with a serumcontent >2% (v/v) is used. If a culture medium is used with a low serumcontent, e.g., 1% (v/v) or a serum-free medium, this step can beomitted. A preferred hydrophobic interaction chromatography medium isbutyl-Sepharose.

The eluate from step (a) or—if used—step (b) is passed overhydroxyapatite in step (c) of the process according to the invention andthe eluate containing EPO is subjected to a concentration step or/and areverse phase HPLC purification step. The concentration is preferablycarried out by exclusion chromatography, such as membrane filtration andthe use of a medium, such as a membrane with an exclusion size of 10 kDhas proven to be favorable.

An isolated human EPO with a specific activity of at least 100,000 U/mgprotein in vivo (normocytaemic mouse) is obtainable by the processaccording to the invention which is free of urinary impurities and mayor may not differ in its glycosylation from recombinant EPO from CHOcells. Preferably, the EPO of the invention has specific activity of atleast 175,000, more preferably at least 200,000, and up to about400-450,000 IU/mg of protein. The human EPO which is obtainable by theprocess according to the invention can contain α-2,3-linked or/andα-2,6-linked sialic acid residues. When EPO obtained from cells whichcontain an endogenously activated EPO gene was examined, the presence ofα-2,3-linked and α-2,6-linked sialic acid residues was found.Furthermore, it was found that human EPO according to the invention hasa content of less than 0.2% N-glycol-neuraminic acid relative to thecontent of N-acetyl neuraminic acid.

The purity of the human EPO obtained in accordance with the invention isat least 90%, more preferably at least 95% and most preferably at least98% relative to the total protein content. The total protein content canbe determined by reverse phase HPLC, e.g., with a Poros R/2H column.

Human species of proteins, such as EPO can be obtained by the processaccording to the invention which differ in their amino acid sequence.Thus for example using mass spectrometric analysis (MALDI-MS) it wasfound that a human EPO can be isolated from HeLa S3 cells wherein theisolated EPO consists essentially of a polypeptide with a length of 165amino acids which is formed by C-terminal processing of an arginineresidue. Up to about 15% of the recovered EPO may be 166 amino acids inlength, depending on the culture conditions used. In addition a humanEPO can also be obtained which consists of a polypeptide with a lengthof 166 amino acids, i.e., a non-processed EPO. Human EPO from Namalwacells was isolated which contained a mixture of polypeptides with alength of 165 and 166 amino acids.

Such proteins, such as human EPO, can be used as an active substance fora pharmaceutical preparation which can optionally contain further activesubstances as well as standard pharmaceutical auxiliary, carrier andadditive substances.

A further aspect of the present invention is an isolated nucleic acidmolecule which codes for a human EPO with a modified sequence in theregion of the first 4 amino acids of the signal peptide as describedsupra.

Genomic DNA and cDNA are both a part of the invention.

How these and other features of the invention are achieved will be seenin the examples which follow.

EXAMPLE 1

1. Culture

Cell lines were seeded at a concentration of 0.1-5×10⁵ per ml,preferably 0.5-2×105 cells per ml in culture plates containing DMEM and10% FCS or RPMI 1640 and 10% FCS and the cell count was determined everytwo to three days during culture with a counting chamber, with orwithout trypsinization, in a medium recommended for the respective cellsand under suitable conditions. Cells which exhibited 16 to 256population doublings, preferably 64 to 128 population doublings, withinone week culture were assessed as positive (+, ++ or +++).

1.2 Ability to Culture in Suspension

In order to determine the ability to culture the cells in suspension,samples were cultured for 14 days at 37° C. and 7% CO₂ while stirringcontinuously in medium as above with and without the addition of serum,e.g., fetal calf serum. Cells which exhibited at least 5 populationdoublings during this phase were assessed as being suitable (+) for asuspension culture.

1.3 Ability to Culture in Serum-free Medium

In order to determine whether the cells could be cultured in serum freemedium, they were cultured under conditions according to 1.1 for 14 daysat a density of 1-10×10⁵ cells/ml in culture vessels in the basic medium(without serum supplementation). Cells whose population doubled at least5 times during this period (determined by cell counting) were assessedas being suitable (+) for serum-free culture.

1.4 Determination of Endogenous Expression of the Target Gene

In order to determine whether the target protein is produced in theselected cells, the cells were seeded at a cell density of 0.01 to 2×10⁶cells/ml preferably 0.5 to 1×10⁶ cells/ml culture medium for 24 hours.The cell culture supernatant was removed later, cells were discarded,and the content of cell protein in the cell culture supernatant wasdetermined by known methods, e.g., by a specific immunoassay for therespective protein.

In the case of EPO the content was determined by means of an ELISA. Forthis, streptavidin-coated microtitre plates were coated withbiotinylated anti-EPO antibodies and incubated with a solutioncontaining protein (1% w/v) to block unspecific binding. Then, 0.1 mlsamples of culture supernatant was added and incubated overnight. Afterwashing, peroxidase-conjugated monoclonal anti-EPO antibodies were addedfor 2 hours. The peroxidase reaction was carried out in a Perkin Elmerphotometer at 405 nm using ABTS as a substrate.

The detection limit for EPO in this test was 10 pg EPO/ml. Cells whichproduced less than 10 pg EPO/ml when seeded at 10⁶ cells/ml wereassessed as non producers and as suitable (+).

1.5 Determination of the Number of Copies of the Target Gene

In order to examine the number of copies of the target gene in the cellline, human genomic DNA was isolated from ca. 10⁸ cells and quantifiedfollowing Sambrook et al., Molecular Cloning, A Laboratory Manual(1989), Cold Spring Harbor Laboratory Press. After cleaving the DNA withrestriction enzymes, e.g., Agel and AscI or BamHI, HindIII and SalI theDNA fragments were separated according to size by agarose gelelectrophoresis and finally transferred onto a nylon membrane andimmobilized.

The immobilized DNA was hybridized with a digoxigenin-labelled DNA probewhich was specific for the locus of the target gene or for thechromosome on which the target gene was located and washed understringent conditions. The specific hybridization signals were detectedwith the aid of standard chemiluminescence methodologies usingradiation-sensitive films.

1.6 Determination of the Nucleic Acid Sequence of the Target Gene

The genomic DNA was isolated from ca. 10⁷ cells using a commerciallyavailable DNA isolation kit.

A pair of PCR primers was used to amplify the target gene. The sequencesof these primers were complementary to sequences which flank the codingregion of the target gene. This enabled the amplification of the entirecoding region of the target gene.

The PCR product was either directly subjected to sequence analysis orcloned into a vector and subsequently sequenced. Sequencing primerswhich are complementary to sequences from the intron regions of thetarget gene were used, so that the sequences of the exon regions of thetarget gene could be obtained completely. The sequencing was carried outon an automated sequencer using commercially available materials andinstructions.

1.7 Determination of the Glycosylation Pattern

In order to determine the glycosylation pattern of EPO, the cell linesto be tested were transfected with the plasmid pEPO 227 which contains a4 kb HindIII/EcoRI fragment of the human EPO gene sequence under thecontrol of the SV40 promoter (Jacobs et al. Nature 313 (1985), 806;Lee-Huang et al. Gene 128 (1993), 272). The cells were transfected inthe presence of lipofectamine using a commercially available reagent kitaccording to the manufacturer's instructions. The EPO content wasdetermined by ELISA in the cell supernatant isolated 2 to 5 days later.

The cell supernatant was concentrated and compared to known EPO productsby isoelectric focusing (Righetti P. G., in: Work T. S., Burdon R. H.(ed.), Isoelectric focusing: Theory, methodology and applications,Elsevier Biomedical Press, Amsterdam (1983)). Human cells which yieldeda comparable glycosylation pattern to known EPO products, e.g., urinaryEPO were assessed as suitable (+).

1.8 Determination of Viral Contamination

1.8.1. Analyses by Means of Cell Culture

In order to determine viral contamination of human cell lines testedlysates of the cells were incubated with a detector cell line in orderto detect cytopathic effects. Hemadsorption analyses were also carriedout.

In order to produce the lysate, a suspension of 10⁶ cells was lysed in 1ml buffer by a rapid freeze-thaw process. The cellular residue wasseparated by centrifugation and the supernatant was added to thedetector cell lines. HepG2 (ATCC HB-8065; Nature 282 (1979), 615-616),MRC-5 (ATCC-1587) and Vero (ATCC CCL-171; Jacobs, Nature 227 (1970),168-170) cells were used as detector cell lines. Polio, SV, andinfluenza type viruses were used as a positive control. Detector celllines that had been cultured without lysate were used as negativecontrol. In order to determine cytopathic effects the detector celllines were regularly examined over a period of at least 14 days.

For hemadsorption analysis, Vero cells which had been incubated with thecell lysates or with the controls were admixed, after 7 days, witherythrocytes from chickens, pigs or humans. An attachment of theerythrocytes to the monolayer of cultured cells indicates viralcontamination of the cultures.

1.8.2. In vivo Analysis

Lysates of the cell lines to be examined were prepared as stated in1.8.1 and injected intraperitoneally or intracerebrally into newbornmice (0.1 ml per injection). The mice were observed with regard tomorbidity and mortality over a period of 14 days.

1.8.3 Specific Detection of Viral Proteins

The presence of specific viral proteins, e.g., Epstein-Barr virusproteins (nuclear protein or capsid antigen) was tested by adding humanserum of EBV-positive bands to immobilized cells of the cell line to betested. The virus antigens were then detected by adding complement andthe corresponding anti-human complement C3 fluorescein conjugate (todetect the nuclear antigen) or via anti-human globulin fluorescein (todetect the capsid antigen).

The human cell lines HepG2, HT 1080, Namalwa, HeLa, and HeLaS3 weretested as described. The results are set forth at Table 1.

It can be seen from table 1 that cell lines HT1080, Namalwa and HeLa S3satisfied the required and preferred criteria with Namalwa and HeLa S3being particularly preferred.

EXAMPLE 2

Cloning of EPO Homology Regions

Homology regions of the EPO gene were amplified by PCR using humanplacenta genomic DNA. For this, two PCR products were prepared from a6.3 kB long homology region from the region of the 5′-untranslatedsequences of the EPO gene, exon 1 and intron 1 (cf FIG. 1). The primersused to prepare the PCR product 1 had the following sequences:5′-CGCGGCGGAT CCCAGGGAGC TGGGTTGACC GG-3′ (SEQ ID NO: 1) and5′-GGCCGCGAAT TCTCCGCGCC TGGCCGGGGT CCCTCAGC-3′ (SEQ ID NO: 2). Theprimers used to prepare the PCR product 2 had the following sequences:5′-CGCGGCGGAT CCTCTCCTCC CTCCCAAGCT GCAATC-3′ (SEQ ID NO: 3) and5′-GGCCGCGAAT TCTAGAACAG ATAGCCAGGC TGAGAG-3′ (SEQ ID NO: 4).

The desired segments were cut out of the PCR products 1 and 2 byrestriction cleavage (PCR product 1: HindIII, PCR product 2: HindIII andEcoRV) and cloned into the vector pCRII which had been cleaved withHindIII and EcoRV. The recombinant vector obtained in this manner wasnamed 5epopcr1000 (cf. FIG. 2).

EXAMPLE 3

Construction of EPO Gene Targeting Vectors

3.1 A gene activation sequence which contains the NEO gene, the DHFRgene and a CMV promoter/enhancer (cf. FIG. 3) was inserted into the AgeIsite of the plasmid 5epocr1000 containing the EPO homology region toobtain the plasmid p176(cf. FIG. 4a). In order to bring the CMV promoteras close as possible to the translation start site of the EPO gene, a963 bp long segment was deleted between the restriction sites AscI andAgeI (partial cleavage) to obtain the plasmid p179 (FIG. 4b).

3.2 In order to optimize expression, nucleotides in exon 1 which codefor the beginning of the EPO leader sequence Met-Gly-Val-His werereplaced by the synthetic sequence Met-Ser-Ala-His. This sequence wasobtained as a template using appropriate primers by amplifying a genomicEPO DNA sequence, e.g., of the plasmid pEPO148 which contains a 3.5 kBBstEII/EcoRI fragment (including exons 1-5) of the human EPO genesequence under the control of the SV40 promoter (Jacobs et al., Nature313 (1985), 806 and Lee-Huang et al., Gene 128 (1993), 227). The plasmidp187 was obtained in this process (FIG. 4b).

3.3 The plasmid p189 was prepared from the plasmid p187 by insertion ofthe Herpes Simplex virus thymidine kinase gene (HSV-TK) which wasderived from Psvtk-1 (PvuII/NarI fragment) (FIG. 4c). The HSV-TK gene isunder the control of the SV40 promoter and the 3′ end of intron 1(EcoRV/ClaI) in the opposite orientation relative to the CMV promoterand should serve to negatively select for homologous recombination.

3.4 For the construction of plasmid p190, a SfiI/BglII fragment ofpHEAVY, a plasmid which contains the cDNA of an arginine mutant of DHFRdescribed by Simonsen et al. (Proc. Natl. Acad. Sci. USA 80 (1983),2495) was subcloned into the plasmid pGenak-1 cleaved with SfiI andBglII. This plasmid contains the NEO gene under the control of the RSVpromoter and the late SV40 polyadenylation site as a terminator, themurine DHFR gene under the control of the early SV40 promoter and theearly SV40 polyadenylation site as a terminator (Kaufmann et al., Mol.Cell. Biol. 2 (1982), 1304; Okayama et al., Mol. Cell. Biol. 3 (1983),280 and Schimke, J. Biol. Chem. 263 (1988), 5989) and the CMV promoter(Boshart et al., Cell 41 (1995), 521). Afterwards an HpaI fragment whichcontained the cDNA coding for the DHFR arginine mutant was ligated intothe plasmid p189 cleaved with HpaI to obtain the plasmid p190 (FIG. 4d).

3.5 In order to obtain a transfection vector without the HSV-TK gene, anAscI/NheI fragment of the plasmid p190 which contained the geneactivation sequence was ligated into the AscI/NheI fragment of theplasmid p187 containing the exon 1. The resulting plasmid was named p192(FIG. 4e).

EXAMPLE 4

Transfection of Cells

Various cell lines were selected for the production of EPO andtransfected with targeting vectors.

4.1 Namalwa Cells

Namalwa cells were cultured in T150 tissue culture flasks andtransfected by electroporation (1×10⁷ cells/800 μl electroporationbuffer 20 mM Hepes, 138 mM NaCl, 5 mM KCl, 0.7 mM Na₂HPO₄, 6 mMD-glucose monohydrate pH 7.0, 10 μg linearized DNA, 960 μF, 260 V BioRadgene pulser). After the electroporation the cells were cultured in RPMI1640, 10% (v/v) fetal calf serum (FCS), 2 mM L-glutamine, 1 mM sodiumpyruvate in forty 96-well plates. After two days the cells were culturedfor 10 to 20 days in medium containing 1 mg/ml G-418. The supernatantwas tested in a solid phase ELISA for the production of EPO as describedsupra. The EPO producing clones were expanded in 24-well plates and T-25tissue culture flasks. Aliquots were frozen and the cells were subclonedby FACS (Ventage, Becton Dickinson). The subclones were repeatedlytested for EPO production.

4.2 HT 1080 Cells

The conditions were as described for the Namalwa cells except that theHT1080 cells were cultured in DMEM, 10% (v/v) FCS, 2 mM L-glutamine, 1mM sodium pyruvate. For transfection by electroporation, cells weredetached from the walls of the culture vessels by trypsinization. Afterelectroporation 1×10⁷ cells were cultured in DMEM, 10% (v/v) FCS, 2 mML-glutamine, 1 mM sodium pyruvate in 5 96-well plates.

4.3 HeLa S3 Cells

Conditions were as described for the Namalwa cells except that the HeLaS3 cells were cultured in RPMI 1640, 10% (v/v) FCS, 2 mM L-glutamine, 1%(v/v) NEM non-essential amino acids, 1 mM sodium pyruvate. For thetransfection by electroporation the cells were detached from the wallsof the culture vessels by trypsinization. The conditions for theelectroporation were 960 μF/250 V. After the electroporation the cellswere cultured in RPMI 1640, 10% (v/v) FCS, 2 mM L-glutamine, 1% (v/v)NEM, 1 mM sodium pyruvate in T75 tissue culture flasks. 24 hours afterelectroporation the cells were trypsinized and cultured for 10 to 15days in a medium containing 600 μg/ml G-418 in 10 96-well plates.

EXAMPLE 5

Selection of EPO Producing Clones

The culture supernatant of transfected cells was tested in an EPO ELISA,as described supra. All steps were carried out at room temperature.96-well plates pre-coated with streptavidin were coated withbiotinylated anti-EPO antibodies. For coating, the plates were firstwashed with 50 mM sodium phosphate pH 7.2, 0.05% (v/v) Tween 20. Then0.01 ml coating buffer (4 μg/ml biotinylated antibody, 10 mM sodiumphosphate pH 7.2, 3 g/l bovine serum albumin, 20 g/l sucrose, 9 g/lNaCl) was added to each well and incubated for 3 hours at roomtemperature. Then the plates were washed with 50 mM sodium phosphate pH7.2, dried and sealed.

Before the test and after washing three times with 0.3 mlphosphate-buffered saline (PBS), 0.05% Tween 20 (Sigma), the plates wereincubated overnight with 0.2 ml PBS, 1% (w/v) protein per well in orderto block unspecific binding.

After removing the blocking solution 0.1 ml culture supernatant wasadded and the plates were incubated overnight. The individual wells wereeach washed three times, with 0.3 ml PBS, 0.05% Tween 20. Then 100 μlperoxidase (POD) conjugated monoclonal anti-EPO antibody was added for 2hours. The wells were each subsequently washed, three times, with 0.3 mlPBS, 0.05% Tween 20. Then the peroxidase reaction was carried out usingABTS as the substrate in a Perkin Elmer photometer at 405 nm. A standardcalibration curve using recombinant EPO from CHO cells was used tocalculate the EPO concentrations.

EXAMPLE 6

EPO Gene Amplification

In order to increase the EPO expression, the EPO producing clones werecultured in the presence of increasing concentrations (100 pM-1000 nM)of methotrexate (MTX). At each MTX concentration the clones were testedby an ELISA (see example 1.4) for the production of EPO. Strongproducers were subcloned by limiting dilution.

EXAMPLE 7

Signal Sequence Mutations

In order to optimize the leader sequence of the EPO molecule, the firstfour amino acids coded by exon 1 were substituted. Primers with varioussequences (SEQ ID NOS: 4-17; the 3′ primer contained a CellI site toselect modified sequences) were used to obtain an AscI/XbaI fragment asa template by PCR using the plasmid pEPO227 which contains a 4 kBHindIII/EcoRI fragment (including exons 1-5) of the human EPO genesequence under the control of the SV40 promoter (Jacobs et al., Nature313 (1985), 806; Lee-Huang et al., Gene 128 (1993), 227). The resultingfragments were subsequently cloned into the plasmid pEPO148 (example3.2) to obtain the plasmids pEPO 182, 183, 184 and 185 (FIG. 5). The EPOgene expression was driven by an SV40 promoter. COS-7 cells weretransiently transfected with the constructs (DEAE-dextan method) and thecells were tested for EPO production 48 hours after the transfection.

The mutated leader sequence Met-Ser-Ala-His obtained in this manner withthe best EPO expression was used to construct gene targeting vectors(cf. example 3.2).

EXAMPLE 8

Characterization of Cell Lines Producing EPO

Three different cell lines (Namalwa, HeLa S3 and HT 1080) were selectedfor EPO gene activation. EPO producing clones were obtained bytransfection with the plasmids p179, p187, p189, p190 or p192, describedsupra.

About 160,000 NEO resistant clones were tested for EPO production, ofwhich 12-15 secreted EPO reproducibly into the cell supernatant insignificant yield.

Of these clones it was surprisingly possible to identify a total of 7EPO clones which produced EPO in adequate amounts for a large-scaleproduction without gene amplification by MTX. This is a surprisinglyhigh yield. The EPO production of these clones was in the range of frommore than about 200 ng/ml up to more than about 1000 ng/ml/10⁶ cells/24hours.

After gene amplification with 500 nM MTX it was possible to increase theEPO production of the identified EPO clones to more than about 3000ng/ml/10⁶ cells/24 hours. Further increase of the MTX concentration to1000 nM led to production of more than about 7000 ng/ml/10⁶ cells/24hours.

The clones obtained produced EPO even under serum-free cultureconditions.

EXAMPLE 9

Characterization of the Genome of the EPO Producing Clones 9.1 Methods

Human genomic DNA was isolated from ca. 10⁸ cells and quantified,following Sambrook et al., supra. After cleavage of the genomic DNA withrestriction enzymes, e.g., AgeI and AscI or BamHI, HindIII and SalI, theDNA fragments were separated according to their size by agarose gelelectrophoresis and subsequently transferred and immobilized on a nylonmembrane.

The immobilized DNA was hybridized with digoxigenin-labelledEPO-specific or gene activation sequence-specific DNA probes and washedunder stringent conditions. The specific hybridization signals weredetected with the aid of a chemiluminescent method using radiationsensitive films.

9.2 Results

The treatment of cells with 500 nM MTX led to an amplification of thehybridization signal in the EPO locus by a factor of 5 to 10. When itwas increased further to 1000 nM MTX, amplification of >10 was obtained(FIG. 6a).

In the case of hybridization with an EPO-specific probe, the copies ofthe chromosome 7 which were not affected by homologous recombinationwere also detected. As can be seen in FIG. 6b, these DNA fragments whichalso hybridize have a different size that is clearly distinguishable andtheir signal strength was not changed by the use of MTX.

EXAMPLE 10

Purification of EPO from Culture Supernatants of Human Cell Lines (HeLaS3; Namalwa and HT1080)

Two methods were used to purify EPO from cell culture supernatants ofhuman cell lines. These differed in the number and principle of thechromatography steps and were used depending on the composition of themedium and the EPO concentrations. In these experiments, EPO waspurified from cell free supernatant of cultured HeLaS3 cells, where thecell culture medium included 2% (v/v) fetal calf serum.

Method 1:

1st step: blue Sepharose column

2nd step: butyl Sepharose column

3rd step: hydroxyapatite column

4th step: concentration

Method 2:

1st step: blue Sepharose column

2nd step: hydroxyapatite column

3rd step: concentration

(alternative 3rd step: RP-HPLC)

1. Blue Sepharose Column

A 5 ml blue Sepharose containing column was equilibrated with at least 5column volumes (CV) buffer A (20 mM Tris-HCl, pH 7.0; 5 mM CaCl₂; 100 mMNaCl). Subsequently, 70 ml HeLa S3 cell supernatant (containing ca. 245μg EPO and 70-100 mg total protein) was absorbed overnight at a flowrate of 0.5 ml/min in a circulation process.

The column was washed with at least 5 CVs buffer B (20 mM Tris-HCl, pH7.0; 5 mM CaCl₂; 250 mM NaCl) and at least 5 CVs buffer C (20 mMTris-HCl, pH 7.0; 0.2 mM CaCl₂, 250 mM NaCl) at 0.5 ml/min. The successof the washing was monitored by measuring the protein content at OD280.

EPO was eluted with buffer D (100 mM Tris-HCl, pH 7.0; 0.2 mM CaCl₂; 2 MNaCl) at a flow rate of 0.5 ml/min. The elution solution was collectedin 1-2 ml fractions.

The EPO content of the fractions, the wash solutions and the flowthrough were determined by reverse phase (RP)-HPLC by applying analiquot to a column. Alternatively an immunological dot-blot was carriedout for the qualitative identification of fractions containing EPO.

Fractions containing EPO (8-12 ml) were pooled and applied to abutyl-Sepharose column.

The yield after the blue Sepharose column was ca. 175 μg EPO(corresponds to ca. 70%). In general the yield after blue Sepharose wasbetween 50-75%.

2. Butyl Sepharose Column (Hydrophobic Interaction Chromatography)

A 2-3 ml butyl Sepharose column was prepared, equilibrated with at least5 CV buffer D (100 mM Tris-HCl, pH 7.0; 0.2 mM CaCl₂; 2 M NaCl), andsubsequently the blue Sepharose pool containing EPO from 1, supra (ca.150 μg EPO) was absorbed at a flow rate of 0.5 ml/min.

The column was washed with at least 5 CV buffer E (20 mM Tris-HCl, pH7.0; 2 M NaCl and 10% isopropanol) at 0.5 ml/min. The success of thewashing was monitored by measuring the protein content at OD280.

EPO was eluted with buffer F (20 mM Tris-HCl, pH 7.0; 2 M NaCl and 20%isopropanol) at a flow rate of 0.5 ml/min. The elution solution wascollected in 1-2 ml fractions.

The EPO content of the fractions, the wash solutions and the flowthrough were determined by RP-HPLC by applying an aliquot to a POROSR2/H column. Alternatively, an immunological dot-blot was carried outfor the qualitative identification of fractions containing EPO.

Fractions containing EPO (10-15 ml) were pooled and applied to ahydroxyapatite column.

The yield after the butyl Sepharose column was ca. 130 μg EPO(corresponds to ca. 85%). In general the yield of the butyl Sepharosewas between 60-85% of the applied blue Sepharose pool.

3. Hydroxyapatite Column

A 5 ml hydroxyapatite column was equilibrated with at least 5 CV bufferF (20 mM Tris-HCl, pH 7.0; 2 M NaCl; 20% isopropanol) and subsequentlythe butyl Sepharose pool containing EPO from 2, supra (ca. 125 μg EPO)was absorbed at a flow rate of 0.5 ml/min.

The column was washed with at least 5 CV buffer G (20 mM Tris-HCl, pH7.0; 2 M NaCl) at 0.5 ml/min. The success of the washing was monitoredby measuring the protein content at OD280.

EPO was eluted with buffer H (10 mM Na-phosphate, pH 7.0; 80 mM NaCl) ata flow rate of 0.5 ml/min. The elution solution was collected in 1-2 mlfractions.

The EPO content of the fractions, the wash solutions and the eluant weredetermined by RP-HPLC by applying an aliquot to a POROS R2/H column.

Fractions containing EPO (3-6 ml) were pooled. The yield of thehydroxyapatite column was ca. 80 μg EPO (corresponds to ca. 60%). Ingeneral the yield of the hydroxyapatite column was between 50-65% of theapplied butyl Sepharose pool.

4. Concentration

The pooled EPO fractions from the hydroxyapatite step were concentratedin centrifugation units with an exclusion size of 10 kD to aconcentration of 0.1-0.5 mg/ml, admixed with 0.01% Tween 20 and storedin aliquots at −20° C.

Yield Table: EPO (μg) Yield (%) Initial 245 100 blue Sepharose 175 70butyl Sepharose column 130 53 hydroxyapatite column 80 33 concentration60 25

The purity of the isolated EPO was about >90%, usually even >95%.

Method 2 was also used to increase the EPO yield in which the butylSepharose step was omitted. This method can be applied above all to cellculture supernatants without or with the addition of 1% (v/v) FCS andyields isolated EPO of approximately the same purity (90-95%).

The presence of 5 mM CaCl₂ in the equilibration buffer (buffer F) forthe hydroxyapatite column led to improved binding in this method andthus also to reproducible elution behavior of EPO in the hydroxyapatitestep. Hence, method 2 was carried out with the following buffers usingbasically the same process as method 1:

1. Blue Sepharose Column:

equilibration buffer (buffer A):

20 mM Tris-HCl, pH 7.0;

5 mM CaCl₂; 100 mM NaCl

wash buffer 1 (buffer B):

20 mM Tris-HCl, pH 7.0;

5 mM CaCl₂; 250 mM NaCl

wash buffer 2 (buffer C):

20 mM Tris-HCl, pH 7.0;

5 mM CaCl₂, 250 mM NaCl

Elution buffer (buffer D):

100 mM Tris-HCl, pH 7.0;

5 mM CaCl₂; 2 M NaCl

2. Hydroxyapatite column

equilibration buffer (buffer F):

50 mM Tris-HCl, pH 7.0;

5 mM CaCl₂; 1 M NaCl

wash buffer (buffer G):

10 mM Tris-HCl, pH 7.0;

5 mM CaCl₂; 80 mM NaCl

elution buffer (buffer H):

10 mM Na phosphate, pH 7.0;

0.5 mM CaCl₂; 80 mM NaCl

Yield scheme: EPO (μg) Yield (%) initial 600 100 blue Sepharose 450 75hydroxyapatite column 335 55 concentration 310 52

The addition of 5 mM CaCl₂ in buffers C, D, E, F, and G in method 1 alsoled to improved binding and more defined elution from the hydroxyapatitecolumn.

EXAMPLE 11

Determination of the Specific Activity in vivo of EPO from Human CellLines (Bioassay on the Normocytaemic Mouse)

The dose-dependent activity of EPO on the proliferation anddifferentiation of erythrocyte precursor cells was determined in vivo inmice via the increase of reticulocytes in the blood after administrationof EPO.

For this, groups of eight mice received various doses of the EPO sampleto be analyzed, and of an EPO standard (matched with the EPO WHOstandard). The mice were subsequently kept under constant definedconditions. Four days after administration of EPO, blood was collectedfrom the mice and the reticulocytes were stained with acridine orange.The reticulocyte number per 30,000 erythrocytes was determined bymicrofluorimetry in a flow cytometer by analyzing the red fluorescencehistogram.

The biological activity of the cells was calculated from the values forthe reticulocyte numbers of the sample and of the standard at thevarious doses according to the method described by Linder of pairedquantity determination with parallel lines (A. Linder, “Planen undAuswerten von Versuchen,” 3rd edition, 1969, Birkenhäuser Verlag Basel).

Result: specific activity EPO from the cell line U/mg HeLa S3 (sample 1)100,000 HeLa S3 (sample 2) 110,000

10 1 32 DNA Homo sapiens 1 cgcggcggat cccagggagc tgggttgacc gg 32 2 38DNA Homo Sapiens 2 ggccgcgaat tctccgcgcc tggccggggt ccctcagc 38 3 36 DNAHomo Sapiens 3 cgccgcggat cctctcctcc ctcccaagct gcaatc 36 4 36 DNA HomoSapiens 4 ggccgcgaat tctagaacag atagccaggc tgagag 36 5 4 PRT HomoSapiens Variant The first Xaa is Gly or Ser. The second Xaa is Ala, 5Met Xaa Xaa Xaa 1 6 4 PRT Homo Sapiens 6 Met Gly Val His 1 7 4 PRT HomoSapiens 7 Met Gly Ala His 1 8 4 PRT Homo Sapiens 8 Met Ser Ala His 1 9 4PRT Homo Sapiens 9 Met Gly Val Pro 1 10 4 PRT Homo Sapiens 10 Met SerVal His 1

We claim:
 1. An isolated, human recombinant cell produced by endogenousgene activation which comprises a CMV promoter sequence in operablelinkage with the endogenous gene which encodes a protein, wherein 10⁶ ofsaid cells produce at least 200 ng of said protein per 24 hours, whereinsaid protein is erythropoietin and wherein said endogenous gene is notamplified.
 2. The isolated, human recombinant cell of claim 1, wherein10⁶ of said cells produce from about 200 to about 3000 ng of saidprotein per 24 hours.
 3. An isolated, human recombinant cell produced byendogenous gene activation wherein said human recombinant cell comprisesa heterologous promoter in operable linkage with said endogenous gene,wherein said endogenous gene encodes erythropoietin, wherein said CMVpromoter and said endogenous gene are amplified and wherein 10⁶ of saidcells produce at least 1000 ng of erythropoietin per 24 hours.
 4. Theisolated, human recombinant cell of claim 3, wherein 10⁶ of said cellsproduces from 1000-25,000 ng of said protein per 24 hours.
 5. Theisolated, human recombinant cell of claim 1 or claim 3, wherein saidcell is culturable in serum-free medium.
 6. The isolated, humanrecombinant cell of claim 1 or claim 3, wherein said cell is an HT 1080cell, a HeLa S3 cell, or a Namalwa cell.
 7. A process for purifyingerythropoietin comprising: (i) culturing the isolated human recombinantcell of claim 1 in a culture medium, under conditions favoringproduction of erythropoietin encoded by said endogenous gene, to producea cell supernatant containing erythropoietin, (ii) passing said cellsupernatant over an affinity chromatography medium to isolate a fractioncontaining erythropoietin, (iii) passing the fraction obtained in (ii)over a hydroxyapatite column to isolate a fraction containingerythropoietin, and (iv) concentrating the fraction obtained in (iii)over a reverse phase, high performance liquid chromatography medium toisolate erythropoietin therefrom.
 8. The process of claim 7, furthercomprising passing the fraction obtained in (i) over a hydrophobicinteraction chromatography medium to obtain an erythropoietin containingfraction prior to contacting said fraction to the hydroxyapatite of(iii).
 9. The process of claim 7, wherein said affinity chromatographymedium is blue Sepharose.
 10. The process of claim 8, wherein saidhydrophobic interaction chromatography medium is butyl Sepharose. 11.The process of claim 7, wherein said concentrating comprises exclusionchromatography.
 12. The process of claim 11, comprising excludingmolecules less than 10 kD from those larger than 10 kD.
 13. An isolated,human recombinant cell which comprises a CMV promoter sequence inoperable linkage with an endogenous gene which encodes a protein,wherein 10⁶ of said cell produces at least 200 ng of said protein per 24hours, wherein said endogenous gene is not amplified, wherein theisolated human recombinant cell is produced by endogenous geneactivation of an endogenous gene in a human cell that comprises morethan two chromosomes containing the endogenous gene and wherein thehuman cell does not express the endogenous gene prior to endogenous geneactivation, wherein said human cell undergoes 5 doublings over a periodof 14 days or less in suspension culture, 5 doublings over a maximumperiod of fourteen days in a serum free culture medium and is capable ofcorrectly glycosylating the protein.
 14. An isolated, human recombinantcell which comprises a CMV promoter sequence in operable linkage with anendogenous gene which encodes a protein, wherein 10⁶ of said cellproduces at least 200 ng of said protein per 24 hours, wherein saidendogenous gene is not amplified, wherein the isolated human recombinantcell is produced by endogenous gene activation of an endogenous geneencoding erythropoietin (EPO) in a human cell that comprises more thantwo chromosomes containing the endogenous gene encoding EPO and whereinthe human cell does not express the endogenous gene prior to endogenousgene activation.
 15. An isolated, human recombinant immortalized cellwhich comprises a CMV promoter sequence in operable linkage with anendogenous gene which encodes a protein, wherein 10⁶ of said cellproduces at least 200 ng of said protein per 24 hours, wherein saidendogenous gene is not amplified, wherein the isolated human recombinantimmortalized cell is produced by endogenous gene activation of anendogenous gene in a human immortalized cell that comprises more thantwo chromosomes containing the endogenous gene and wherein the humancell does not express the endogenous gene prior to endogenous geneactivation.